Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Biochemistry. 2013 Dec 3;52(48):8677-86. doi: 10.1021/bi401290j. Epub 2013 Nov 14.
Diol dehydratase-reactivase (DD-R) is a molecular chaperone that reactivates inactivated holodiol dehydratase (DD) by cofactor exchange. Its ADP-bound and ATP-bound forms are high-affinity and low-affinity forms for DD, respectively. Among DD-Rs mutated at the nucleotide-binding site, neither the Dα8N nor Dα413N mutant was effective as a reactivase. Although Dα413N showed ATPase activity, it did not mediate cyanocobalamin (CN-Cbl) release from the DD·CN-Cbl complex in the presence of ATP or ADP and formed a tight complex with apoDD even in the presence of ATP, suggesting the involvement of Aspα413 in the nucleotide switch. In contrast, Dα8N showed very low ATPase activity and did not mediate CN-Cbl release from the complex in the presence of ATP, but it did cause about 50% release in the presence of ADP. The complex formation of this mutant with DD was partially reversed by ATP, suggesting that Aspα8 is involved in the ATPase activity but only partially in the nucleotide switch. Among DD-Rs mutated at the Mg(2+)-binding site, only Eβ31Q was about 30% as active as wild-type DD-R and formed a tight complex with apoDD, indicating that the DD-R β subunit is not absolutely required for reactivation. If subunit swapping occurs between the DD-R β and DD β subunits, Gluβ97 of DD would coordinate to Mg(2+). The complex of Eβ97Q DD with CN-Cbl was not activated by wild-type DD-R. No complex was formed between this mutant and wild-type DD-R, indicating that the coordination of Gluβ97 to Mg(2+) is essential for subunit swapping and therefore for (re)activation.
二醇脱水酶复活酶（DD-R）是一种分子伴侣，通过辅酶交换使失活的全羟二醇脱水酶（DD）复活。其 ADP 结合形式和 ATP 结合形式分别是 DD 的高亲和力和低亲和力形式。在核苷酸结合位点发生突变的 DD-R 中，Dα8N 和 Dα413N 突变体均不能有效地作为复活酶。尽管 Dα413N 表现出 ATP 酶活性，但在存在 ATP 或 ADP 的情况下，它不能介导氰钴胺素（CN-Cbl）从 DD·CN-Cbl 复合物中释放，并且即使在存在 ATP 的情况下也与脱辅基 DD 形成紧密复合物，表明 Aspα413 参与核苷酸开关。相比之下，Dα8N 表现出非常低的 ATP 酶活性，并且在存在 ATP 的情况下不能介导 CN-Cbl 从复合物中释放，但在存在 ADP 的情况下会导致约 50%的释放。该突变体与 DD 的复合物形成部分被 ATP 逆转，表明 Aspα8 参与 ATP 酶活性但仅部分参与核苷酸开关。在 Mg(2+)结合位点发生突变的 DD-R 中，只有 Eβ31Q 的活性约为野生型 DD-R 的 30%，并且与脱辅基 DD 形成紧密复合物，表明 DD-Rβ 亚基并非复活所必需。如果 DD-Rβ 和 DDβ 亚基之间发生亚基交换，DD 的 Gluβ97 将与 Mg(2+)配位。野生型 DD-R 不能激活 Eβ97Q DD 与 CN-Cbl 的复合物。该突变体与野生型 DD-R 之间未形成复合物，表明 Gluβ97 与 Mg(2+)的配位对于亚基交换，进而对于（再）激活是必需的。
